Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure

Abstract

Very little is known about how cellular osmosensors monitor changes in osmolarity of the environment. Here, we report that in yeast, Sln1 osmosensor histidine kinase monitors changes in turgor pressures. Reductions in turgor caused by either hyperosmotic stress, nystatin, or removal of cell wall activate MAPK Hog1 specifically through the SLN1 branch, but not through the SHO1 branch of the high osmolarity glycerol pathway. The integrity of the periplasmic region of Sln1 was essential for its sensor function. We found that activity of the plant histidine kinase cytokinin response 1 (Cre1) is also regulated by changes in turgor pressure, in a manner identical to that of Sln1, in the presence of cytokinin. We propose that Sln1 and Cre1 are turgor sensors, and that similar turgor-sensing mechanisms might regulate hyperosmotic stress responses both in yeast and plants.

Figure 1.

Sln1 and Sho1 have distinct cellular distributions. (A) Architecture of the SLN1 and SHO1 branches of the HOG pathway. (B) Either the SLN1 or SHO1 branch is sufficient to survive on high osmolarity. The wild-type, ste11Δ, ssk2Δ ssk22Δ, or pbs2Δ mutant cells were spotted on low (YPD) and high (YPD + 1.5 M sorbitol) osmolarity media plates, and were grown for 2 d at 30°C. (C) Sln1 has a nearly uniform cytoplasmic membrane distribution. The localization of the Sln1–GFP and the Sho1–GFP fusion proteins was analyzed by fluorescent microscopy in unstressed cells. GFP, fluorescence images; DIC, differential interference contrast images. (D) Hyperosmotic stress induces a clustering of Sln1. Wild-type or pbs2Δ mutant cells expressing Sln1–GFP were observed by fluorescent microscopy before and after (5 min) addition of 0.4 M NaCl.

Figure 1.

Sln1 and Sho1 have distinct cellular distributions. (A) Architecture of the SLN1 and SHO1 branches of the HOG pathway. (B) Either the SLN1 or SHO1 branch is sufficient to survive on high osmolarity. The wild-type, ste11Δ, ssk2Δ ssk22Δ, or pbs2Δ mutant cells were spotted on low (YPD) and high (YPD + 1.5 M sorbitol) osmolarity media plates, and were grown for 2 d at 30°C. (C) Sln1 has a nearly uniform cytoplasmic membrane distribution. The localization of the Sln1–GFP and the Sho1–GFP fusion proteins was analyzed by fluorescent microscopy in unstressed cells. GFP, fluorescence images; DIC, differential interference contrast images. (D) Hyperosmotic stress induces a clustering of Sln1. Wild-type or pbs2Δ mutant cells expressing Sln1–GFP were observed by fluorescent microscopy before and after (5 min) addition of 0.4 M NaCl.

Figure 2.

The SLN1 branch of the HOG pathway is stimulated by turgor reduction. (A) The actin cytoskeleton dynamics does not affect the HOG pathway regulation. The ste11Δ or ssk2Δ ssk22Δ mutant cells were preincubated for 30 min in the presence of latrunculin-A (LAT-A, 100 μM), jasplakinolide (JAS, 10 μM), or the vehicle DMSO, and the Hog1 phosphorylation was assayed before and after (5 min) addition of 0.4 M NaCl. In each strain, the multi-drug resistance gene PDR5 was deleted to increase drug uptake. (B) The activation of Hog1 correlates with turgor-dependent cell volume shrinkage. The wild-type cells were exposed to cycles of high (YPD + 0.4 M NaCl) and low (YPD) osmolarity media in 2-min intervals. For each cycle, samples were withdrawn to determine the Hog1 phosphorylation and the relative cell volume. The wild-type cells treated with nystatin (10 μM, 5 min) were analyzed similarly. (C) Nystatin activates the SLN1 branch of the HOG pathway. The phosphorylation of Hog1 was determined in the mutant strains after treatment with nystatin (NYS; 10 μM, 5 min). (D) Time course of Hog1 activation by nystatin. Wild-type cells were treated with 0.4 M NaCl or 10 μM nystatin (NYS), and the Hog1 phosphorylation was analyzed. (E and F) Treatment by nystatin or removal of cell wall stimulates the SLN1 branch of the HOG pathway. The wild-type, ste11Δ, or ssk2Δ ssk22Δ cells were treated with nystatin (E; +NYS, 10 μM) or zymolyase (F; +zymo). The control samples (−NYS and −zymo) were prepared identically, except the nystatin or zymolyase addition. The samples were analyzed for the Hog1 phosphorylation directly or after (0.4 M NaCl, 5 min) osmotic stress.

Figure 3.

Plant histidine kinase Cre1 responds to changes in turgor pressure. (A) The active (zeatin-bound) Cre1 histidine kinase is inhibited by high osmolarity stress. The double mutant sln1Δ ste11Δ GAL1-PTP2 (strain BVRY179) expressing plasmid encoding for SLN1 (sln1Δ SLN1), CRE1 (sln1Δ ADH1-CRE1 or sln1Δ CYC1-CRE1), or empty vector (sln1Δ vec), was grown in glucose media (see Materials and methods for the experimental details) in the presence (+zea) or absence (−zea) of zeatin. The activity of Hog1 was then determined in these strains before and after (0.4 M NaCl, 5 min) high osmolarity stress. (B) The time course of Hog1 activation by NaCl or sorbitol in cells expressing Cre1. The Hog1 activity was analyzed in the sln1Δ ste11Δ GAL1-PTP2 strain (BVRY179) carrying a plasmid encoding SLN1 or CRE1 or vector alone (vec). These strains were grown in glucose media (as in A) supplemented with 10 μM zeatin and stressed by 0.4 M NaCl or by 1.0 M sorbitol for indicated times. (C) Cre1 activity responds to reduction in turgor pressure. The sln1Δ ste11Δ GAL1-PTP2 strain (BVRY179) expressing SLN1 or CRE1 was incubated with sorbitol (sorb; 1.0 M, 5 min), nystatin (NYS; 10 μM, 5 min), or zymolyase (zymo; as in Fig. 2 F) before the samples were assayed for Hog1 phosphorylation. HeLa cells were treated with 1 M sorbitol for 15 min before cell extracts were prepared to determine p38 MAPK activation by immunoblot analysis using an anti-phospho-p38 antibody.

Figure 4.

Integrity of extracellular domain is essential for Sln1 function. (A) Deletion of aa 138–150 in the periplasmic domain impairs the Sln1 function. The culture of the sln1Δ GAL1-PTP2 strain (TM182) grown in galactose media and carrying centromeric plasmid encoding for wild-type SLN1 or the mutant SLN1 with a deletion of aa 138–150 (sln1Δ9) was dropped onto media containing galactose (GAL) or glucose (GLC). (B) Absence of N-linked glycosylation is not responsible for the phenotype of the sln1Δ9 mutant. The sln1N138, 142Q mutant was tested for the complementation of the sln1Δ strain (TM182) as described in A. (C) The Sln1Δ9–GFP fusion protein has abnormal cellular distribution. The localization of Sln1–GFP and Sln1Δ9–GFP expressed from centromeric plasmid was determined in the sln1Δ strain (TM182) by fluorescent microscopy. (D and E) The phenotype of sln1Δ9 is suppressed by VPS10 overexpression. VPS10 was identified as a multicopy suppressor gene of the sln1Δ9 mutant (tested as in A). (E) The protein levels of HA-tagged Sln1 or Sln1Δ9 were compared by immunoblot analysis in the sln1Δ strain (TM182) transformed with a VPS10 multicopy plasmid or the empty vector (D). (F) The periplasmic domains of Sln1 and Cre1 are interchangeable. The culture of the sln1Δ ste11Δ GAL1-PTP2 strain (BVRY179) transformed with the SLN1 variant containing the periplasmic domain from Cre1 (SLN1-CRE1ecto) was analyzed for Hog1 phosphorylation before and after osmotic stress (0.4 M NaCl) in the absence or presence of zeatin in glucose media as in Fig. 3 A. (G) Domain swapping analysis between Sln1 and Cre1. The centromeric plasmids encoding Sln1–Cre1 hybrids were tested for the complementation of the sln1Δ ste11Δ GAL1-PTP2 strain (BVRY179) as described in A.